Treating severe acute respiratory syndrome

ABSTRACT

Severe acute respiratory syndrome is treated with a natural human alpha interferon, a dsRNA or both natural human alpha interferon and a dsRNA.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of Ser. No. 10/842,474filed May 11, 2004, now abandoned which in turn claims priority fromU.S. provisional patent application Ser. No. 60/470,893 filed May 16,2003 and Ser. No. 60/517,882 flied Nov. 7, 2003.

Procedures are provided for combating the effects of coronavirus-inducedconditions by the administration of an α-interferon composed of amixture of naturally occurring α-interferons or a synthetic,specifically configured, double-stranded ribonucleic acid (dsRNA) orboth an α-interferon and a dsRNA.

BACKGROUND

Severe Acute Respiratory Syndrome (SARS) is a new disease that israpidly spreading within China and other countries around the world.Although, a combination of ribavirin, a synthetic,non-interferon-inducing, broad spectrum antiviral nucleoside, andcorticosteroids is commonly used as therapy, especially in China,laboratory testing by the National Institutes of Health (NIH) foundribavirin to have no effect on this coronavirus. This lack of efficacysuggests the need for an effective therapeutic regimen.

DESCRIPTION OF THE INVENTION

Described is use of an α-interferon, preferably a natural, multi-speciesα-interferon in the treatment of the symptoms associated with SARS inpatients including human patients infected with the SARS virus, alsoreferred to as the SARS-associated coronavirus (SARS-CoV).Alternatively, a dsRNA may be used in the treatment of the symptomsassociated with SARS-associated coronavirus in patents including humanpatients infected with the SARS-associated coronavirus. Also describedis the coordinated use of both (1) an α-interferon, preferably anatural, multi-species α-interferon and conjointly therewith (2) a dsRNAin the treatment of the symptoms associated with SARS-associatedcoronavirus in patients including human patients infected theSARS-associated coronavirus. Procedures for attaining a favorabletherapeutic and clinical result and compositions for accomplishing thesame are described. Preferably the dsRNA is administered with theα-interferon and preferably the dsRNA is rI_(n).r(C₁₂U)_(n.), PolyA.Poly U or rI_(n).r(C₂₉,G)_(n), in which r is ribo.

In the context of the present invention, what is meant by “coordinated”use is, independently, either (i) co-administration, i.e. substantiallysimultaneous or sequential administration of the α-interferon and of thedsRNA, or (ii) the administration of a composition comprising theα-interferon and the dsRNA in combination and in a mixture, in additionto optional pharmaceutically acceptable excipients and/or vehicles.

For internal administration the α-interferon may, for example, beformulated in conventional manner for oral or rectal administration.Formulations for oral administration include aqueous solutions, syrups,elixirs, powders, granules, tablets and capsules which typically containconventional excipients such as binding agents, fillers, lubricants,disintegrants, wetting agents, suspending agents, emulsifying agents,preservatives, buffer salts, flavoring, coloring and/or sweeteningagents.

The α-interferon component of the therapeutic procedures is preferablyAlferon N Injection® the only approved natural, multi-species,α-interferon available in the United States. It is the first naturalsource, multi-species interferon and is a consistent mixture of at leastseven species of α-interferon. In contrast, the other availableα-interferons are single molecular species of α-interferon made inbacteria using DNA recombinant technology. These single molecularspecies of α-interferon also lack an important structural carbohydratecomponent because this glycosylation step is not performed during thebacterial process.

Unlike species of α-interferon produced by recombinant techniques,Alferon N Injection® is produced by human white blood cells which areable to glycosylate the multiple α-interferon species. Reverse PhaseHPLC studies show that Alferon N Injection® is a consistent mixture ofat least seven species of alpha interferon (α2, α4, α7, α8, α10, α16,α7). This natural-source interferon has unique anti-viral propertiesdistinguishing it from genetically engineered interferons. The highpurity of Alferon N Injection® and its advantage as a natural mixture ofseven interferon species, some of which, like species 8b, have greaterantiviral activities than other species, for example, species 2b, whichis the only component of Intron A. The superior antiviral activities forexample in the treatment of chronic hepatitis C virus (HCV) and (HIV)and tolerability of Alferon N Injection® compared to other availablerecombinant interferons, such as Intron A and Roferon A, have beenreported.

It is reported Alferon N Injection® has activity against a naturalcoronavirus infection in pigs. Transmissible gastroenteritis (TGE)coronavirus causes an acute gastroenteritis in swine. The diarrhea anddehydration caused by this viral infection result in a high mortalityrate in neonates with severity inversely related to the age of theanimal. In fact, in piglets less than 14 days of age themorality/morbidity rate typically approaches 100%. Piglets, ages 1-12days treated with 1.0, 10.0, or 20.0 IU of Alferon N Injection® werefound to have an increased survival compared to the control groupindicating benefit of this natural mixture of α-interferons in combatingthis particular coronavirus.

The invention includes methods of enhancing therapy againstcoronaviruses by administering to patients interferons, particularlynatural human alpha interferon and together or conjointly a synthetic,specifically configured, double-stranded ribonucleic acid (dsRNA). ThedsRNA of choice is AMPLIGEN® rintatolimod, a synthetic, specificallyconfigured, double-stranded ribonucleic acid (dsRNA) which retains theimmunostimulatory and antiviral properties of other double-stranded RNAmolecules (dsRNA) but exhibits greatly reduced toxicity. Like otherdsRNA, AMPLIGEN® rintatolimod can elicit the induction of interferon andother cytokines. AMPLIGEN® rintatolimod has the ability to stimulate avariety of dsRNA-dependent intracellular antiviral defense mechanismsincluding the 2′,5′-oligoadenylate synthetase/RNase L and protein kinaseenzyme pathways.

The mismatched dsRNA may be of the general formula rI_(n).r(C₁₂U)_(n).In this and the other formulae that follow r=ribo. Other mismatcheddsRNAs for use in the present invention are based on copolynucleotidesselected from poly (C_(m),U) and poly (C_(m)G) in which m is an integerhaving a value of from 4 to 29 and are mismatched analogs of complexesof polyriboinosinic and polyribocytidilic acids, formed by modifyingrI_(n).rC_(n) to incorporate unpaired bases (uracil or guanine) alongthe polyribocytidylate (rC_(m)) strand. Alternatively, the dsRNA may bederived from r(I).r(C) dsRNA by modifying the ribosyl backbone ofpolyriboinosinic acid (rI_(n)), e.g., by including 2′-O-methyl ribosylresidues. The mismatched may be complexed with an RNA-stabilizingpolymer such as lysine cellulose. Of these mismatched analogs ofrI_(n).rC_(n), the preferred ones are of the general formularI_(n).r(C₁₁₋₁₄,U)_(n.) or rI_(n).r(C₂₉,G)_(n), and are described byCarter and Ts'o in U.S. Pat. Nos. 4,130,641 and 4,024,222 thedisclosures of which are hereby incorporated by reference. The dsRNA'sdescribed therein generally are suitable for use according to thepresent invention.

Other examples of mismatched dsRNA for use in the invention include:

r(I).r(C₄, U)

r(I).r(C₇, U)

r(I).r(C₁₃, U)

r(I).r(C₂₂, U)

r(I).r(C₂₀, G) and

r(I).r(C_(p.23), G_(>p)).

Alternatively the dsRNA may be the matched form, thus polyadenylic acidcomplexed with polyuridylic acid (poly A.poly U) may also be used.

When administered 24 hours prior to viral challenge, ampligen has beendemonstrated in viral cytopathic inhibition assays and neutral redassays to inhibit human coronavirus strain OC-43, thus suggestingprotective activity of ampligen against human chromavirus prior to anencounter with this virus.

α-interferon and/or the dsRNA may be administered for therapy by anysuitable route including oral, rectal, nasal, topical (includingtransdermal, buccal and sublingual), vaginal and parenteral (includingsubcutaneous, intramuscular, intravenous intradermal, and intravitreal).It will be appreciated that the preferred route will vary with thecondition and age of the recipient, the nature of the infection and thechosen active ingredient.

As indicated above, severe acute respiratory syndrome (SARS) is causedby a newly identified member of the coronavirus family. AMPLIGEN®rintatolimod a double-stranded RNA (dsRNA), is reported to exhibitantiviral activity against the coronavirus, Mouse Hepatitis Virus Type-3(MHV-3) see Hepatology 3:837, 1983. MHV-3 is a coronavirus which causesboth a fulminant and a chronic form of hepatitis depending on the mousestrain studied. AMPLIGEN® rintatolimod treatment had a positive effectagainst the MHV-3 coronavirus in both mouse models. In an acuteinfection model, Balb/cJ mice exposed to MHV-3 and then treated twicewith AMPLIGEN® rintatolimod survived up to four times longer thanuntreated mice. Since no treatment was given beyond 24 hourspost-exposure, it is likely that additional AMPLIGEN® rintatolimodtreatments would have had an even greater impact on survival. In thechronic hepatitis model, C3H mice treated after exposure to the MHV-3coronavirus cleared the virus quickly and did not develop chronichepatitis. Thus, AMPLIGEN® rintatolimod has shown activity against thecoronavirus, MHV-3, in two different mouse models, increasing survivalin the acute infection model and completely abrogating the infection inthe chronic model.

In a further study it has been determined Alferon® inhibits SARS-CoV ata high specific activity in Vero 76 cells (African green monkey) inculture. Alferon® is a highly purified natural a-interferon obtainedfrom human leucocytes and consists of seven different α-interferon aminoacid sequences α2, α4, α7, α8, α10, α16, and α17). Inhibition wasquantitated by visual cytopathic effect (CPE), inhibition of thecellular uptake of the vital dye, neutral red (NR), and by virus yieldreduction. NR assay was conducted immediately following visual assay.Fifty-percent effective doses (EC5O) were calculated for both CPE and NRassays by regression analysis. Quantitative values for viral yieldreduction assays were expressed as 90% effective concentrations (EC9O),representing the drug concentration required to reduce SARS-CoV titersby one log₁₀ and were calculated by regression analysis. Vein cellsinfected at a MOI of 0.001 visually exhibited 100% CPE over a 3-5 dayincubation period without treatment. Alferon® inhibited SARS-CoV at anECSO=5,696+/−1,703 (SEM) IU/ml (visual) and 10,740+/−5,161 (SEM) IU/ml(NR). Viral load reduction by one log₁₀ was 78,000+/−22,000 (SEM) IU/ml.

The beneficial effects of Alferon® are also reported by Tan et al,Emerging Infectious Diseases—www.cdc.gov/eid—Vol. 10 No. 4, April 2004,in which several commercially available, clinically approved compoundsfrom several antiviral pharmacologic classes screened to determine thepresence of in vitro anti-SARS-CoV activity. Of the 18 antiviralcompounds tested Alferon® was found to be the most active FDA-approvedinterferon when tested against the SARS coronavirus. This contrasts withthe recombinant α-interferons, Roferon and Intron A, which were notfound to be active against the SARS coronavirus when tested at muchhigher concentrations.

It is also reported the activity of interferon can potentially beamplified by the addition of a double-stranded RNA drug, AMPLIGEN®rintatolimod. While interferon up-regulates certain intracellularantiviral pathways, dsRNAs, like AMPLIGEN® rintatolimod, are required tofully activate these important antiviral pathways. When interferons arecombine with AMPLIGEN® rintatolimod synergistic antiviral and antitumoreffects are seen. Moreover, AMPLIGEN® rintatolimod has already shownstrong antiviral activity in two separate animal models of thecoronavirus (MHV-3). Although uncertainty now exists regarding thecharacteristics of this coronavirus, in the event it is determined theSARS-associated coronavirus elaborates IFN neutralizing products,AMPLIGEN® rintatolimod has potential to override these inhibitors andachieve an antiviral effect.

In addition, as a further attribute of the dsRNA arm of the disclosedtherapeutic combination therapy, synergistic antiviral and antitumoreffects have been demonstrated using AMPLIGEN® rintatolimod treatment incombination with all three types of interferon (α, β and γ). Thesesynergistic effects have been seen against HIV and a variety ofdifferent histologic tumor types. Four human tumor cell lines werestudied for their response to antiproliferative effects of AMPLIGEN®rintatolimod in combination with various interferons. Results indicatethat (1) AMPLIGEN® rintatolimod worked synergistically with allinterferons in all cell lines studied; (2) growth inhibition of cellsresistant to interferons can be potentiated by low doses of AMPLIGEN®rintatolimod; (3) the antiproliferative effect of interferons can bepotentiated by AMPLIGEN® rintatolimod in AMPLIGEN®rintatolimod-resistant cells; and (4) AMPLIGEN® rintatolimod works by amechanism(s) other than, or in addition to, the induction of interferon.See Montefiori, AIDS Res. and Human Retroviruses 5:193-203, 1989 andHubbell, Int. J. Cancer 37:359-365, 1986.

The recommended dosage of the components will depend on the clinicalstatus of the patient and the experience of the clinician in treatingsimilar infection. As a general guideline dosage of Alferon N Injection®utilized for systemic infections is 5 to 10 million units (sq) thriceweekly. The AMPLIGEN® rintatolimod dose schedule is 400 mg by IVinfusion twice weekly, although these amounts and/or dosage frequencymay be varied by the clinician in response to the patient's condition.The components may be administered at the same time, for instance asmixture of the α-interferon and dsRNA, independently as the α-interferonthen the dsRNA or the α-interferon and the dsRNA may be administered ina time-spaced manner.

EXAMPLE

Effects of Alferon N®, an alfa-n3 human interferon, on the replicationof SARSCoV in vitro.

Vero 76 cells (African green monkey kidney) were obtained from AmericanType Culture Collection (Manassas, Va.). The growth medium was Eagle'sminimum essential medium with non-essential amino acids (MEM), 5% FBSand 0.1% NaHCO₃. The test medium was MEM supplemented with 2% FBS, 0.18%NaHCO₃ and 50 μg gentamicin/ml. The SARS coronavirus, strain 200300592(Urbani), was obtained from James Comer (Centers for Disease Control,Atlanta, Ga.). Human leukocyte derived interferon alfa-n3 (03-6600) waskindly provided by Hemispherx Biopharma, Inc. (New Brunswick, N.J.) as astock solution of 5×10⁶ units/ml.

Using cytopathic effect (CPE) reduction assays read visually andverified spectrophotometrically by neutral red (NR) uptake assay of thesame plate (Barnard et al., 2001), an interferon alfa-n3 was evaluatedfor anti-SARSCoV activity in Vera 76 cells.

Virus at a multiplicity of infection of 0.001 was added to 96 wellplates seeded with near confluent monolayers of cells in which drug hadbeen serially diluted, using 10-fold or ½ log dilution series. Additionof virus was within five minutes after exposure of cells to drug.

The cells were incubated at 37° C. until the untreated virus controlsdisplayed destruction of the monolayers (100% CPE, 3-5 days). The plateswere then scored for cytotoxicity and viral CPE by microscopicexamination, usually followed immediately by neutral red staining andprocessing for spectrophotometric reading. EC50 values (theconcentration of compound needed to inhibit the cytopathic effect to 50%of the control value) and IC50 values (the concentration at which uptakeof neutral red or cytotoxic effects was reduced by 50% compared tocontrol cells) was calculated by regression analysis. Values wereexpressed as mean±the standard error of the mean. For the visual assay,n=10 and for the NR assay, n=5. A selective index (IC50/EC5O) for eachcompound was then calculated. Compounds found active by these assayswere then further evaluated for inhibitory activity in two separatevirus yield reduction assays (Barnard et al., 2001). EC9O values werederived by regression analysis from those assays and represent theconcentration at which virus yields were reduced by 1 log₁₀. The EC9Ovalues were averaged and the average was expressed as the mean the ±standard error of the mean.

It was found Alferon N® inhibited SARSCoV, with an EC5O=5,696±1703 IU byvisual CPE inhibition assay and an EC5O 10,740±5,161 IU/ml by NR assay.However, viral cytopathic effects (CPE) were readily apparent at alldilutions tested, although at higher compound doses, CPE was greatlyreduced compared to the virus replication controls (data not shown).This phenomenon was verified by virus yield reduction assay in whicheach dilution of drug was sampled and quantified for the presence ofsurviving virus or newly produced virus. At each dilution of compound,infectious virus was detected, with lower amounts of virus beingdetected at the higher concentrations of compound (data not shown). Theconcentration at which virus load was reduced by 1 log 10 (EC9O) 78000±22,000 IU/ml.

From these studies it was determined Alferon N® worked well in reducingvirus cytopathic effect, with an EC5O of 5,696±1703 IU/ml.

Cinatl J, Morgenstern B, Bauer G, Chandra P, Rabenau H, Doerr H W.Treatment of SARS with human interferons. Lancet 2003; 362:293-4.

Barnard D L, Stowell V D, Seley K L, Hegde V R, Das S R, Rajappan V P,et al. Inhibition of measles virus replication by 5′-nor carbocyclicadenosine analogues. Antiviral Chem Chemother 2001; 12:241-250.

1. A method of treating severe acute respiratory syndrome comprisingadministering to an infected subject a dsRNA; wherein the dsRNA isrI_(n).r(C₁₁₋₁₄,U)_(n), Poly A.Poly U, or rI_(n).r(C₂₉,G)_(n) in which nis an integer and r is ribo.
 2. A method of treating severe acuterespiratory syndrome comprising the coordinated administration to aninfected subject of (1) a natural human alpha interferon and (2) adsRNA: wherein the dsRNA is rI_(n).r(C₁₁₋₁₄,U)_(n), Poly A.Poly U, orrI.r(C₂₉,G)_(n) in which n is an integer and r is ribo.
 3. The methodaccording to claim 1, wherein the dsRNA is rI_(n).r(C₁₂U)_(n).
 4. Themethod according to claim 2, wherein the dsRNA is rI_(n).r(C₁₂U)_(n). 5.The method according to claim 2, wherein the interferon is administeredorally.
 6. The method according to claim 2, wherein the interferon isadministered intravenously.
 7. The method according to claim 2, whereinthe interferon is administered intramuscularly.
 8. The method accordingto claim 2, wherein the interferon is administered subcutaneously. 9.The method according to claim 4, wherein the interferon is administeredorally.
 10. The method according to claim 4, wherein the interferon isadministered intravenously.
 11. The method according to claim 4, whereinthe interferon is administered intramuscularly.
 12. The method accordingto claim 4, wherein the interferon is administered subcutaneously.